Abstract. Multi-parametric analysis by flow cytometry has become an essential tool to characterize complex lymphocyte populations. The combination of multiple fluorescent markers hasenabled strategies to define functional cell subsets, especiallyimportant in characterizing disease etiology and progression. Following guidelines provided by the FITMaN initiative on immunophenotyping (sponsored by FOCIS Centers of Excellence), two 10-color panels were developed using a “backbone” strategy: 8-color panels sharing common reagents with allowance for 2 elective reagents. This approach allows modification of the panels with relative ease. The cocktail development process took into account the selected antigenic immunophenotypes, the characteristics of available fluorochromes, and the use of well-defined control populations.

Results. Data from case studies are presented here with special emphasis on optimization of flow analysis and compensation visualization. A standard set of gating strategy can be applied to both panels while generating reproducible and optimal characterization of CD4+CD25+ Treg subsets with respect to additional differentiation/functional surface markers on 5 normal donors.

Conclusions. This FOCIS FITMaN design approach aims to create a standardized testing condition for optimized resolution of Treg populations. Our data demonstrates the ability of our designed panels to achieve our aims and can be incorporated to enhance capabilities for clinical studies and to render assays compatible across platforms for correlative analyses.

Materials and MethodsAntibody conjugates were obtained and titered for optimal test dosages, which spanned from 1.25 µL to 20 µL. All dye-conjugated antibodies were acquired from Beckman Coulter (Brea, CA), with the exceptions of: CD39-FITC, CCR4-PE and CCR7-PE, which were obtained from a third party.100 µL of normal, whole blood was stained with the appropriate volumes for single colors and nine color cocktails. Samples wereprepared using VersaLyseTM Lysing Solution after 20 minute incubation at room temperature in the dark. Samples were then centrifuged, washed, recentrifuged, and resupsended in 7-Aminoactinomycin D (7-AAD). After a 20-minute incubation at room temperature, cells were fixed in 750 µL of buffer + 0.5% formaldehyde. Samples were assessed on the GalliosTM cytometer (Beckman Coulter), and analyzed using Kaluza software (Beckman Coulter).

Results

Table 2. Rationale for FOCIS Panel. Final design is shown above, which fulfills all given requirements. In addition, this design incorporates a backbone strategy that would only require one compensation matrix for both panels. As a result, one gating strategy for CD4+/CD4+CD25+ is applicable for both panels. CD39 is placed in the FITC channel as an option to enhance finer resolution in differentiating T/ T-regulatory cells. The compensation matrix for the two panels is derived from single color controls and is shown here.

Requirement

• 2 panels• Live/Dead • CCR4/CCR7 on PE• CD38 on APC

Additional design

BackboneEase of compensation

Figure 1. Preliminary Gating Strategy. Live cells are gated from dead cells using 7AAD staining. From this gate, the lymphocyte subpopulation is identified and from it singlets are selected. CD3 positive cells are gated on from the live/lymph/singlets and then further discriminated into CD4 and CD8 subsets. CD4 positive cells in panel 1 are further resolved by plotting CD127 versus CD25 to identify two CD4+CD25+ subsets. For panel 2, CD4 positive cells are plotted against CD25 to identify the T-regulatory and non T-regulatory cells. A FMO (fluorescence- minus-one) stain is also performed without CD25 to verify that the CD25 positive cells are correctly gated.

7AAD

FS

SS

FS

FS INT

FSTOF

(Peak width)

FMO

FULL STAIN

CD4 KrO

CD8APCA700

CD3 ECD

CD4KrO

CD4 KrO

PC7

CD4 KrO

CD25PC7

FMO

FULL STAIN

CD25 PC7

CD127APC

PC7

CD127APC

Panel 1 Panel 2

CCR4PE

CD45RA APCA750

CCR4PE

CD39 FITC

CD4+CD25- CD4+CD25+ Pop. 1 (CD127-)

CD4+CD25+ Pop. 2 (CD127 low)

CCR4PE

CD39 FITC

CCR4PE

CD39 FITC

CD127APC

CD25 PC7

CD127APC

CD25 PC7

FSINT

SS INT

FSINT

SS INT

CD4+CD25+ Pop. 2

CD4+CD25+ Pop. 1

Figure 2. Finer characterization of Treg (CD4+CD25+CD127+/-) cells from panel 1. Comparison of bivariate Density plots from non -Tregulatory cells (CD4+CD25-), CD4+CD25+ population 1 (CD127-) and population 2 (CD127+) are shown in the upper panels. All three populations can be further separated into 3 subsets based CCR4 and CD39 expression. The comparison of these 3 subsets from the two CD4+CD25+ populations is shown in the bottom panels. Finally, the CD4+CD25+ subsets are shown in radar plots in the bottom right section to reveal their occupancy in multi-parametric dimensions.

CD39+CCR4+ (Pop. 2)

CD39-CCR4- (Pop. 2) CD4+CD25+CD127- (Pop. 1)

CD39-CCR4+ (Pop. 2)

CCR7 PE

CD45RA APCA750

CCR7 PE

CD39 FITC

CD45RAAPCA750

CD38 APC

CD4+CD25-

CD4+CD25+

CCR7 PE

CD45RA APCA750

CCR7 PE

CD39 FITC

CD45RAAPCA750

CD38 APC

CD39FITC

CD45RA APCA750

CD38APC

CD39 FITC

CD45RAAPCA750

HLADR PB

CD39FITC

CD45RA APCA750

CD38APC

CD39 FITC

CD45RAAPCA750

HLADR PB

CD38APC

CD39 FITC

CD4+CD25+

Naive Central Memory Effector Memory

CD38APC

CD39 FITC

Figure 3. Finer characterization of Treg (CD4+CD25+CD127+/-) cells from panel 2. CD4+CD25+ and CD4+CD25- cells are further divided into 4 distinct subsets: Naïve, Central memory, Effector memory, and Effector cells. Each of these subsets is color-coded and can be followed when these cells are analyzed in other parameters, see upper section. Shown in the bottom left section, the three main subsets in CD4+CD25+ Treg cells have distinct CD39 and CD38 patterns in their phenotypic progression.Both CD4+CD25+ and CD4+CD25- populations are shown in radar plots in the bottom right section to reveal their occupancy in multi-parametric dimensions.

CD4+CD25+ CD4+CD25-

Table 3. Gating hierarchy and statistics for FOCIS FITMan panels. The hierarchal scheme and population percentages for donor 1 in both panels are shown in the left section. The statistical analysis of all five donors normalized to singlets is shown in the right section.

Panel 1: Hierarchal Gating and Population Statistics-Donor 1

Panel 2: Hierarchal Gating and Population Statistics-Donor 1

Percentages of Singlets

Summary• We have created a set of panel proposals that fulfills the FOCIS FITMan design requirements.

• The panel proposal is designed in a manner that one compensation matrix will satisfy both panels and also an initial gating strategy that can be applied to both panels.

• We have demonstrated the feasibility of these panels to resolve T cell populations and more specifically, to generate detailed characterization of CD4+CD25+ T-regulatory subsets.

• We have shown the applicability of these panels in the analysis of five normal donors.

James W Tung, Jonel Lawson, Carlos A Garcia, Jeffrey R Cobb, Yetty Irwan, William Godfrey, Michael Reed, Enrique RabellinoBeckman Coulter Inc, Cellular Analysis Business Group, Scientific Affairs, Miami FL

Designing multi-color panels for T/Treg populations using FITMaNguidelines: a case study.

Table1. Antibody conjugates used in study